Project Summary: Dental caries is an infectious disease affecting approximately 90% of adults worldwide. Late stages of caries affect the dental pulp, leading to tissue necrosis and ultimately requiring root canal therapy. Typically, root canals in permanent teeth are treated by removing the necrotic tissue and replacing it with an artificial material. Regenerative endodontics has been proposed as an improved treatment option for these conditions. However, without controllable strategies to engineer the pulp vasculature, effective pulp regeneration is virtually impossible. It has been recently demonstrated that a functional vasculature can be engineered by culturing endothelial cells and stem cells from various sources in the correct microenvironmental conditions. However, the precise requirements specific to regenerating the pulp vasculature remain poorly understood. This project will systematically investigate three overlapping aspects that we propose are key determinants to regenerate the pulp vasculature: (1) matrix physical and mechanical properties, (2) composition, and (3) microarchitecture. In aim 1 we will investigate the contributions of different physical and mechanical properties to the ability of human endothelial colony forming cells (ECFCs) and dental pulp stem cells (DPSCs) to form microvascular networks when embedded in hydrogels that can be photo-crosslinked to have their properties systematically adjusted. We will then engineer pulp tissue-constructs that are pre-vascularized with pre-fabricated endothelial microchannels to enhance pulp regeneration in full-length root canals in-vivo. In aim 2 we will develop injectable and photo-curable hydrogels synthesized from the natural matrix of dentin and modified with methacrylates to test the contribution of matrix composition to the regeneration of the pulp vasculature. Further, we will combine these hydrogels with angiogenic components extracted from the dentin matrix and test their regenerative potential in vitro and in vivo. In aim 3 we will fabricate architecturally controlled gradients of ECFC and DPSC paracrine factors using microfluidics techniques to test the contribution of tissue microarchitecture to the formation of the pulp vasculature. We will then mimic the microarchitectures of vascularized dental pulp by 3D bioprinting tissue constructs that reproduce the organization of the native pulp. In the end of this project we expect to have microengineered a 3D vascularized pulp microenvironment that will improve translational approaches for use in regenerative endodontics in adult teeth.